Dr. Yaakov Nahmias is a current Instructor in Surgery and Bioengineering at Harvard Medical School and seeks to establish himself as an independent investigator in the fields of regenerative medicine and BioMEMS. To that end, Dr. Nahmias submits this five years Career Development Award under the sponsorship of Dr. Mehmet Toner, director of the BioMEMS Resource Center and Dr. David Scadden, co- director of the Harvard Stem Cell Institute, both at Harvard Medical School. This award consists of a detailed career development plan which includes: 1) intensive training in microfabrication, 2) academic courses in Harvard and MIT, 3) seminars and lectures, and 4) advisory committee guidance. Research environment includes state-of-the-art facilities at Massachusetts General Hospital consisting of over 7000 sq ft of research and office space with adjacent cleanroom (class 1000), animal, and characterization facilities. The overall goal of this proposal is to elucidate and control the molecular mechanisms (transcription factor networks) underlying the differentiation of stem cells toward the hepatic phenotype. Our specific aims are to: 1) develop and characterize GFP-responsive mouse ES cell lines to major endodermal transcription factors; 2) microfabricate a living cell array for the culture of ES cells, and 3) elucidate and control the transcription factor networks regulating the differentiation of ES cell-derived endoderm toward the hepatic phenotype using soluble signals. We expect to: (a) develop multiple mouse embryonic stem cell lines with GFP- responsive transcription factor elements, (b) elucidate key genetic pathways underlying the differentiation of ES-derived endoderm toward the hepatic phenotype, and (c) provide new avenues to explore the differentiation potential of embryonic stem cells for scientific and clinical applications. One of the major challenges in the field today is the scarcity of functional human hepatocytes as the cells are notoriously difficult to proliferate in vitro. The treatment of liver failure would require around (10)10 hepatocytes. Treatments on this scale necessitate the development of alternative cellular sources that can potentially produce hepatocytes in high yields and large cell numbers.